Leishmaniasis is a major health problem to humans and is caused by the protozoan parasite Leishmania. Depending on the species, Leishmania-induced pathology ranges from self-healing, cutaneous lesions to fatal, visceral diseases. Leishmania express a family of structurally interrelated glycoconjugates, dominated by its surface lipophosphoglycan (LPG), that have critical roles in parasite survival and virulence. The sharing of structural domains among virtually all parasite surface glycoconjugates, however, often leads to imprecision in our understanding of their unique and/or overlapping roles in vivo. Through identification of specific mutants through forward and reverse genetics, and generation of genetically complemented counterparts, we have established a powerful approach enabling us to systematically dissect the biosynthetic pathway of LPG and related glycoconjugates. We plan to focus on mutants defective in a well chosen set of biochemical steps and genes that will allow us to dissect the role of individual glycoconjugates and/or their specific domains in various stages of the infections cycle. The ability of infectious metacyclic parasites to enter, survive and differentiate into amastigotes following ingestion by macrophages will be studied; where possible amastigote initiated infections will be performed as well, as glycoconjugate function can differ greatly in the two infectious stages. The ability of metacyclic promastigotes or amastigotes to induce pathology in susceptible and resistant mice will be studied, and long term persistence evaluated. Lastly, in collaboration with David Sacks we will evaluate the ability of promastigotes fed within a blood meal to survive and develop within the sand fly vector. Several pathways identified show potential for chemotherapy and/or vaccination strategies in the future. Our ultimate goal is a comprehensive understanding of the genes and gene products responsible for synthesizing Leishmania surface and secreted molecules, and their individual and specific roles in parasite virulence. The four specific aims of this competing renewal application are: 1. To identify candidate glycoconjugate(s) that are key virulence molecules responsible for amastigote stage virulence in L. major, as defined by studies of the avirulent, persistent lpg2- mutant. 2. To characterize new families of mannosyl-phosphate transferases and galactosyltransferases involved in synthesis of the phosphoglycan (PG) repeating unit Gal(21,4)Man(11)-PO4- backbone. 3. To determine the role of the emerging sphingolipid (SL) pathway and inositolphosphorylceramide (IPC) in amastigotes. 4. To develop comprehensive Leishmania glycomics. As part of these studies we plan to test whether the recently discovered active RNAi pathway of L. braziliensis may be productively incorporated into the study of Leishmania glycoconjugates.Project Narrative Leishmania are important tropical parasites, causing disease in more than 10 million people worldwide; more than 400 million people are at risk for infection in endemic regions. US military personnel have significant risk of infection in these areas as well. Depending on the species, Leishmania-induced pathology ranges from self-healing, cutaneous lesions to fatal, visceral diseases. Currently, there are no vaccines available against leishmaniasis, and the only approved chemotherapies are marginally effective, difficult to administer, and have significant associated toxicities. The underlying tenet of our research program is that improved understanding of key pathways required for parasite virulence and viability may provide opportunities for the development of improved therapies. Leishmania express a family of structurally interrelated glycoconjugates, dominated by its surface lipophosphoglycan (LPG), that have critical roles in parasite survival and virulence. The sharing of structural domains among virtually all parasite surface glycoconjugates, however, complicates our understanding of their unique and/or overlapping roles in vivo. To overcome this, we use genetic approaches to make parasite mutants altered in specific molecules, or domains, or smaller substitutions. As there are many possible steps, and some of their effects may be similar, we try to choose ones that will give us the greatest information. Then, we test each mutant in the parasite infectious cycle. Leishmania are normally transmitted by the bite of an infected sand fly, so the first step in infection is the deposition of infective metacyclic form parasites into the skin where they are taken up by macrophages. There they resist host defenses and differentiate into another form called amastigotes, which are adapted for replication and go on to cause disease. Eventually sand flies bite infected animals, and the parasite has to survive within the alimentary tract of the fly. We have good assays for testing the effect of each mutant in each of the steps throughout the infectious cycle. We have good success in previous work, and several pathways and molecules already identified show potential for chemotherapy and/or vaccination strategies in the future. Our ultimate goal is a comprehensive understanding of the genes and gene products responsible for synthesizing Leishmania surface and secreted molecules, and their individual and specific roles in parasite virulence.